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Cytokinins, which are central regulators of cell division and differentiation in plants, are adenine derivatives carrying an isopentenyl side chain that may be hydroxylated. Plants have two classes of isopentenyltransferases (IPTs) acting on the adenine moiety: ATP/ ADP isopentenyltransferases (in Arabidopsis thaliana, AtlPT1, 3, 4-8) and tRNA IPTs (in Arabidopsis, AtlPT2 and 9). ATPJADP IPTs are likely to be responsible for the bulk of cytokinin synthesis, whereas it is thought that cis-zeatin (cZ)-type cytokinins are produced possibly by degradation of cis-hydroxy isopentenyl tRNAs, which are formed by tRNA IPTs. However, these routes are largely hypothetical because of lack of in vivo evidence, because the critical experiment necessary to verify these routes, namely the production and analysis of mutants lacking AtlPTs, has not yet been described. We isolated null mutants for all members of the ATP/ADP IPT and tRNA IPT gene families in Arabidopsis. Notably, our work demonstrates that the atipt1 3 5 7 quadruple mutant possesses severely decreased levels of isopentenyladenine and trans-zeatin (tZ), and their corresponding ribosides, ribotides, and glucosides, and is retarded in its growth. In contrast, these mutants possessed increased levels of cZ-type cytokinins. The atipt2 9 double mutant, on the other hand, lacked isopentenyl- and cis-hydroxy isopentenyl-tRNA, and cZ-type cytokinins. These results indicate that whereas ATP/ADP IPTs are responsible for the bulk of isopentenyladenine- and tZ-type cytokinin synthesis, tRNA IPTs are required for cZ-type cytokinin production. This work clarifies the long-standing questions of the biosynthetic routes for isopentenyladenine-, tZ-, and cZ-type cytokinin production.

Cytokinins are plant hormones with profound roles in growth and development, including control of leaf longevity. Although the cytokinin signal is known to be perceived by histidine kinase receptors, the underlying molecular mechanism and specificity of the receptors leading to delayed leaf senescence have not yet been elucidated. Here, we found that AHK3, one of the three cytokinin receptors in Arabidopsis, plays a major role in controlling cytokinin-mediated leaf longevity through a specific phosphorylation of a response regulator, ARR2. This result was obtained through identification of a gain-of-function Arabidopsis mutant that shows delayed leaf senescence because of a missense mutation in the extracellular domain of AHK3. A loss-of-function mutation in AHK3, but not of the other cytokinin receptors, conferred a reduced sensitivity to cytokinin in cytokinin-dependent delay of leaf senescence and abolished cytokinin-dependent phosphorylation of ARR2. Consistently, transgenic overexpression of wild-type, but not an unphosphorylatable mutant ARR2, led to delayed senescence of leaves. histidine kinase 3 | cytokinin | leaf senescence

Since their discovery as cell-division factors in plant tissue culture about five decades ago, cytokinins have been hypothesized to play a central role in the regulation of cell division and differentiation in plants. To test this hypothesis in planta, we isolated Arabidopsis plants lacking one, two, or three of the genes encoding a subfamily of histidine kinases (CRE1, AHK2, and AHK3) that function as cytokinin receptors. Seeds were obtained for homozygous plants containing mutations in all seven genotypes, namely single, double, and triple mutants, and the responses of germinated seedlings in various cytokinin assays were compared. Both redundant and specific functions for the three different cytokinin receptors were observed. Plants carrying mutations in all three genes did not show cytokinin responses, including inhibition of root elongation, inhibition of foot formation, cell proliferation in and greening of calli, and induction of cytokinin primary-response genes. The triple mutants were small and infertile, with a reduction in meristem size and activity, yet they possessed basic organs: roots, stems, and leaves. These results confirm that cytokinins are a pivotal class of plant growth regulators but provide no evidence that cytokinins are required for the processes of gametogenesis and embryogenesis.

One of the most long-lived models in plant science is the belief that the long-distance transport and ratio of two plant hormones, auxin and cytokinin, at the site of action control major developmental events such as apical dominance. We have used in vivo deuterium labeling and mass spectrometry to investigate the dynamics of homeostatic cross talk between the two plant hormones. Interestingly, auxin mediates a very rapid negative control of the cytokinin pool by mainly suppressing the biosynthesis via the isopenteny-ladenosine-5'-monophosphate-independent pathway. In contrast, the effect of cytokinin overproduction on the entire auxin pool in the plant was slower, indicating that this most likely is mediated through altered development. In addition, we were able to confirm that the lateral root meristems are likely to be the main sites of isopentenyladenosine-5'-monophosphate-dependent cytokinin synthesis, and that the aerial tissue of the plant surprisingly also was a significant source of cytokinin biosynthesis. Our demonstration of shoot-localized synthesis, together with data demonstrating that auxin imposes a very rapid regulation of cytokinin biosynthesis, illustrates that the two hormones can interact also on the metabolic level in controlling plant development, and that the aerial part of the plant has the capacity to synthesize its own cytokinin independent of long-range transport from the root system.

We identified a set of cytokinin-insensitive mutants by using a screen based on the ethylene-mediated triple response observed after treatment with low levels of cytokinins. One group of these mutants disrupts ACS5, a member of the Arabidopsis gene family that encodes 1-aminocyclopropane-1-carboxylate synthase, the first enzyme in ethylene biosynthesis. The ACS5 isoform is mainly responsible for the sustained rise in ethylene biosynthesis observed in response to low levels of cytokinin and appears to be regulated primarily by a posttranscriptional mechanism. Furthermore, the dominant ethylene-overproducing mutant eto2 was found to be the result of an alteration of the carboxy terminus of ACS5, suggesting that this domain acts as a negative regulator of ACS5 function.

Assembly and mutual proximities of [Alpha], [Beta], and [[Gamma].sub.c] subunits of the interleukin 2 receptors (IL-2R) in plasma membranes of Kit 225 K6 T lymphoma cells were investigated by fluorescence resonance energy transfer (FRET) using fluorescein isothiocyanate- and Cy3-conjugated monoclonal antibodies (mAbs) that were directed against the IL-2R[Alpha], IL-2R[Beta], and [[Gamma].sub.c] subunits of IL-2R. The cell-surface distribution of subunits was analyzed at the nanometer scale (2-10 nm) by FRET on a cell-by-cell basis. The cells were probed in resting phase and after coculture with saturating concentrations of IL-2, IL-7, and IL-15. FRET data from donor- and acceptor-labeled IL-2R[Beta]-[Alpha], [Gamma]-[Alpha], and [Gamma]-[Beta] pairs demonstrated close proximity of all subunits to each other in the plasma membrane of resting T cells. These mutual proximities do not appear to represent mAb-induced micro-aggregation, because FRET measurements with Fab fragments of the mAbs gave similar results. The relative proximities were meaningfully modulated by binding of IL-2, IL-7, and IL-15. based on FRET analysis the topology of the three subunits at the surface of resting cells can be best described by a 'triangular model' in the absence of added interleukins. IL-2 strengthens the bridges between the subunits, making the triangle more compact. IL-7 and IL-15 act in the opposite direction by opening the triangle possibly because they associate their private specific [Alpha] receptors with the [Beta] and/or [[Gamma].sub.c] subunits of the IL-2R complex. These data suggest that IL-2R subunits are already colocalized in resting T cells and do not require cytokine-induced redistribution. This colocalization is significantly modulated by binding of relevant interleukins in a cytokine-specific manner.

Study of the expression motifs of a cDNA clone, which encodes an auxin-induced gene, extracted from Zinnia plants, reveals the gene to participate in initial vascular differentiation processes. Cambial region localization and auxin- and cytokinin-controlled-stimulation are the primary features of this gene. The vascular cambium induces the proliferation of secondary phloem and xylem in plants.

Cytokinins mediate light and nutrient signals which stimulate the accumulation of wpk4 mRNA. The gene wpk4, which encoded a potential protein kinase from wheat, is a member of the SNF1 kinase subfamily. Cytokinins induce cell division and chloroplast formation and inhibit apical dominance and sencsence in plants.

Cytokinins are a class of plant-specific hormones that play a central role during the cell cycle and influence numerous developmental programs. Because of the lack of biosynthetic and signaling mutants, the regulatory roles of cytokinins are not well understood. We genetically engineered cytokinin oxidase expression in transgenic tobacco plants to reduce their endogenous cytokinin content. Cytokinin-deficient plants developed stunted shoots with smaller apical meristems. The plastochrone was prolonged, and leaf cell production was only 3-4% that of wild type, indicating an absolute requirement of cytokinins for leaf growth. In contrast, root meristems of transgenic plants were enlarged and gave rise to faster growing and more branched roots. These results suggest that cytokinins are an important regulatory factor of plant meristem activity and morphogenesis, with opposing roles in shoots and roots.